Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.     

Burr medic (Medicago polymorpha L.) selections for improved N2 fixation with naturalised soil rhizobia

Burr medic (Medicago polymorpha L.) is an annual pasture legume that is widely distributed in southern Australian farming systems. Burr medic is nodulated by rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae) that reside in many Australian soils, but the symbioses that develop are often sub-optimal in their rate of N₂ fixation. We attempted to identify burr medic lines, which are able to form effective symbioses with the naturalised Sinorhizobium in Australian field soils, as potential parents for a breeding program. There were three glasshouse experiments. Initially, 222 lines (including the M. polymorpha cvv. Santiago, Serena and Circle Valley) were inoculated with extracts of two soils that had been collected near Waikerie (soil S109) and Lochiel (soil S142) in South Australia. These soils were used because they contained numerically large communities of naturalised Sinorhizobium spp. that produced sub-optimal rates of N₂ fixation with cv. Santiago. None of the 222 lines of burr medic were able to form an effective symbiosis with the rhizobia from soil S109. However, when nodulated by the rhizobia from soil S142, some lines (e.g. SA8194) formed a very effective symbiosis, producing up to double the shoot dry matter (DM) of Santiago and eight times the DM of uninoculated plants. Seven promising lines were selected for further testing (with extracts of nine soils). Subsequently, two lines (SA20056 and SA8194) were selected and their symbiotic performance compared with that of Santiago, using extracts from 28 soils. While soil treatment had a major effect on mean shoot DM (soil N103=120 mg, soil N105=17 mg), the three medic lines performed similarly. Santiago, SA20056 and SA8914 all formed ineffective symbioses with the rhizobia in at least half of the 28 soils, even though >95% of the plants were nodulated. These experiments confirm that ineffective symbioses are common between burr medics and the rhizobia that have become naturalised in many Australian soils. Although some lines of burr medic were identified that were able to form more effective symbioses with the rhizobia in individual soils, none were able to form effective symbioses with a wide range of soil rhizobia. If a plant breeding approach is to be used to improve symbiotic performance of burr medic we propose that its hybridisation with other medic species, that have less specific rhizobial needs, will be required.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

Alleviation of drought stress in the common bean (Phaseolus vulgaris L.) by co-inoculation with Paenibacillus polymyxa and Rhizobium tropici

A greenhouse experiment was performed to evaluate the influence of Rhizobium when co-inoculated with each of two Paenibacillus polymyxa strains, singly and in mixture on growth, nitrogen content, phytohormone levels and nodulation of the common bean (Phaseolus vulgaris L.) under three levels of drought stress. Stress was applied continuously by the control of matric potential (ψ_m) through a porous cup. Bean plants cv. Tenderlake were grown in pots with Fluvic Neosol eutrophic soil under three different ψ_m (S₁ −7.0; S₂ −70.0 and S₃ < −85 kPa). The seeds were inoculated with Rhizobium tropici (CIAT 899) and each of P. polymyxa (DSM 36) and P. polymyxa Loutit (L) singly and in mixture (CIAT 899 + DSM36 + Loutit). Co-inoculation of bean with Rhizobium and both Paenibacillus strains resulted increased plant growth, nitrogen content and nodulation compared to inoculation with Rhizobium alone. This was particularly evident at the most negative ψ_m (S₃ < −85 kPa) we used. Drought stress triggered a change in phytohormonal balance, including an increase in leaf abscisic acid (ABA) content, a small decline in indole acetic acid (IAA) and gibberellic acid (GA₃) and a sharp fall in zeatin content in bean leaves. The content of endogenous Cks decreased under water stress, possibly amplifying the response of shoots to increasing ABA content. We hypothesize that co-inoculation of bean with R. tropici (CIAT 899) and P. polymyxa strains (DSM 36) and Loutit (L) mitigates some of the negative effects of drought stress on bean.     

Elevation of atmospheric CO2 and N-nutritional status modify nodulation, nodule-carbon supply, and root exudation of Phaseolus vulgaris L.

Increased root exudation and a related stimulation of rhizosphere-microbial growth have been hypothesised as possible explanations for a lower nitrogen- (N-) nutritional status of plants grown under elevated atmospheric CO₂ concentrations, due to enhanced plant-microbial N competition in the rhizosphere. Leguminous plants may be able to counterbalance the enhanced N requirement by increased symbiotic N₂ fixation. Only limited information is available about the factors determining the stimulation of symbiotic N₂ fixation in response to elevated CO₂.In this study, short-term effects of elevated CO₂ on quality and quantity of root exudation, and on carbon supply to the nodules were assessed in Phaseolus vulgaris, grown in soil culture with limited (30 mg N kg⁻¹ soil) and sufficient N supply (200 mg N kg⁻¹ soil), at ambient (400 μmol mol⁻¹) and elevated (800 μmol mol⁻¹) atmospheric CO₂ concentrations.Elevated CO₂ reduced N tissue concentrations in both N treatments, accelerated the expression of N deficiency symptoms in the N-limited variant, but did not affect plant biomass production. ¹⁴CO₂ pulse-chase labelling revealed no indication for a general increase in root exudation with subsequent stimulation of rhizosphere microbial growth, resulting in increased N-competition in the rhizosphere at elevated CO₂. However, a CO₂-induced stimulation in root exudation of sugars and malate as a chemo-attractant for rhizobia was detected in 0.5-1.5 cm apical root zones as potential infection sites. Particularly in nodules, elevated CO₂ increased the accumulation of malate as a major carbon source for the microsymbiont and of malonate with essential functions for nodule development. Nodule number, biomass and the proportion of leghaemoglobin-producing nodules were also enhanced. The release of nod-gene-inducing flavonoids (genistein, daidzein and coumestrol) was stimulated under elevated CO₂, independent of the N supply, and was already detectable at early stages of seedling development at 6 days after sowing.     

Isotopic fractionation during N2 fixation by four tropical legumes

To quantify the contribution of biological nitrogen fixation (BNF) to legume crops using the ¹⁵N natural abundance technique, it is necessary to determine the ¹⁵N abundance of the N derived from BNF—the B value. In this study, we used a technique to determine B whereby both legume and non-N₂-fixing reference plants were grown under the same conditions in two similar soils, one artificially labelled with ¹⁵N, and the other not. The proportion of N derived from BNF (%Ndfa) was determined from the plants grown in the ¹⁵N-labelled soil and it was assumed that the %Ndfa values of the legumes grown in the two soils were the same, hence the B value of the legumes could be calculated. The legumes used were velvet bean (Mucuna pruriens), sunnhemp (Crotalaria juncea), groundnut (Arachis hypogaea) and soybean (Glycine max) inoculated, or not, with different strains of rhizobium. The values of %Ndfa were all over 89%, and all the legumes grown in unlabelled soil showed negative δ¹⁵N values even though the plant-available N in this soil was found to be approximately +6.0‰. The B values for the shoot tissue (B_s) were calculated and ranged from approximately −1.4‰ for inoculated sunnhemp and groundnut to −2.4 and −4.5‰ for soybean inoculated with Bradyrhizobium japonicum strain CPAC 7 and Bradyrhizobium elkanii strain 29W, respectively. The B (B_wp) values for the whole plants including roots, nodules and the original seed N were still significantly different between the soybean plants inoculated with CPAC 7 (−1.33‰) and 29W (−2.25‰). In a parallel experiment conducted in monoxenic culture using the same soybean variety and Bradyrhizobium strains, the plants accumulated less N from BNF and the values were less negative, but still significantly different for soybean inoculated with the two different Bradyrhizobium strains. The results suggest that the technique utilized in this study to determine B with legume plants grown in soil in the open air, yields B values that are more appropriate for use under field conditions.     

Arbuscular mycorrhizae affect the N and C economy of nodulated Phaseolus vulgaris (L.) during NH4 nutrition

In the symbiosis between nodulated legume roots and arbuscular mycorrhizal (AM) fungi, the C and N economy can be influenced by the source of N-supply from either AM-derived NH₄⁺ uptake or nodule-derived biological nitrogen fixation (BNF). This relationship was investigated in terms of NH₄⁺ supply and BNF by the two symbionts. Nodulated Phaseolus vulgaris seedlings with and without AM, were hydroponically grown with either 0 N or 1 mM NH₄⁺ supply. Plants were harvested at 30 days after emergence and measurements were taken for biomass, N₂ fixation, photosynthesis, CO₂ and O₂ root respiration, calculated C and N economy. AM roots had higher NH₄⁺ uptake and this was associated with the suppression of BNF and nodule growth. The higher NH₄⁺ uptake in AM roots occurred with lower root maintenance respiration, compared to when N was derived from BNF. There was also an increase in the below-ground sink strength of NH₄⁺ fed AM roots compared to NH₄⁺ fed non-AM roots, as evidenced by the increases in root CO₂ and O₂ respiration and photosynthetic stimulation. These results indicate that although the AM root had higher total below-ground respiratory costs during NH₄⁺ nutrition, there were lower respiratory C costs associated with N derived from AM symbionts in comparison to N from BNF.     

Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)

Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slow-release nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC-MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems.     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.     

Rhizobial inoculation improves drought tolerance,biomass and grain yields of common bean (Phaseolus vulgaris L.) and soybean (Glycine max L.) at Halaba and Boricha in Southern Ethiopia

ABSTRACT

While pulses are staple food-legumes in Ethiopia, their productivity is low due to low soil fertility. Elite rhizobial strains that significantly increased shoot dry weight and nitrogen (N) contents of common beans and soybeans in greenhouse were selected for two-year field trials to evaluate their effect on yields of the pulses in the field. Each pulse had six treatments, namely four rhizobial inoculants, uninoculated control, and synthetic N fertilizer. In the drought-affected year 2015, inoculated pulses tolerated moisture stress better than non-inoculated controls. Inoculation was conducive to higher or equivalent yields compared to synthetic N fertilizer. At Halaba, bean inoculated with strain HAMBI3562 gave the highest grain yield (1500 ± 81 kg ha^?1; mean±SE) while the control yielded only 653 ± 22 kg ha^?1. At Boricha, HAMBI3570 gave a grain yield (640 ± 35 kg ha^?1) comparable to synthetic N. When rainfall was optimal in 2016, inoculation with HAMBI3562 and HAMBI3570 gave grain yields (around 4300 kg ha^?1) equivalent to synthetic N. With soybean, strain HAMBI3513 produced consistently higher or comparable biomass and grain yields compared to synthetic N. In conclusion, HAMBI3562 and HAMBI3570 for beans and HAMBI3513 for soybeans can serve as inoculants for areas having similar conditions as the test areas.     

The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

For optimum production, the use of commercial rhizobial inoculant on pea (Pisum sativum L.) at seeding is necessary in the absence of compatible rhizobial strains or when rhizobial soil populations are low or symbiotically ineffective. Multiple site experiments were conducted to characterize the abundance and effectiveness of resident populations of Rhizobium leguminosarum bv. viciae (Rlv) in eastern Canadian prairie soils. A survey of 20 sites across a broad geographical range of southern Manitoba was carried out in 1998 and was followed by more intensive study of five of the sites in 1999 and 2000. Appreciable nodulation of uninoculated pea was observed at all sites which had previously grown inoculated pea. However, uninoculated pea grown at two sites, which had not previously grown pea, had negligible nodulation. Likewise, wild Lathyrus sp. and Vicia sp. plants collected from uncultivated areas adjacent to agricultural sites were poorly nodulated. In the more intensively studied sites, there was a tendency towards higher nodulation in pea plants receiving commercial inoculant containing Rlv strain PBC108 across all site-years (e.g., 4.7% in nodulation and 22% in nodule mass), but the effect was significant at only 2 of 10 site-years. Despite a relatively high range of soil pH (6-8), regression analysis indicated that decreasing soil pH resulted in lower nodulation rates. Likewise, electrical conductivity (EC) was correlated to nodulation levels, however the effect of EC was likely more indicative of the influence of soil texture and organic matter than salinity. As with nodulation, commercial inoculation tended to increase above-ground dry matter (DM) and fixed-N (estimated by the difference method) at the early pod-filling stage, but again the effects were significant at only 2 of 10 site-years. Specifically, above-ground DM and fixed-N levels were up to 29 and 51% greater, respectively, in inoculated compared to non-inoculated treatments at these sites. Addition of N-fertilizer at a rate of 100 kg N ha⁻¹ decreased nodulation at almost all site-years (by as much as 70% at one site), but rarely resulted in increases in above-ground DM compared to inoculated plots. The study indicates for the first time that populations of infective, and generally effective strains of Rlv occur broadly in agricultural soils across the eastern Canadian prairie, but that there is a tendency for increased symbiotic efficiency with the use of commercial inoculant.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

Genetic diversity analysis of common bean (Phaseolus vulgaris L.) genotypes for resistance to Mexican bean weevil (Zabrotes subfasciatus), using single nucleotide polymorphism and phenotypic markers

ABSTRACT

The objective of this study was to examine the genetic diversity present among 297 common bean genotypes using 2554 SNPs and 12 insects and seed-related traits. The phenotyping was done under laboratory condition while the genotyping was conducted by using the Illumina SNP BeadChip. High phenotypic diversity among traits were recorded, ranging from 0.87 to 0.96, with a mean of 0.92. Principal component and discriminant analyses identi?ed four PCs and three discriminant functions, which explained 82% and 100% of the total phenotypic variations among genotypes, respectively. Polymorphic Information Content ranged from 0.21 to 0.38, with a mean of 0.34. The mean gene diversity among genotypes ranged from 0.24 to 0.50, with a mean of 0.44. Genetic distance ranged from 0.19 to 0.82, with a mean of 0.62, while the phenotypic distance ranged from 0.00 to 1.00, with a mean of 0.64 were observed aamong genotypes. The analysis of molecular variance revealed highly signi?cant differences (p<0.001) among and within individuals and among populations. Both the SNP and the phenotypic markers grouped the 297 genotypes into two major distinct clusters and three sub-clusters. This information is useful for identi?cation and development of common bean germplasm with economically valuable traits and the conservation and utilization of genotypes.     

Host-rhizobia interaction for effective inoculation: evaluation of the potential use of the ureide assay to monitor the symbiotic performance of tepary bean (Phaseolus acutifolius A. Gray)

Domesticated and wild-type tepary beans (Phaseolus acutifolius A. Gray) were grown with or without inoculation with rhizobia in pots under bacteriologically controlled conditions in a temperature-controlled glasshouse. Seeds were inoculated with a mixture of seven strains isolated from nodules collected from domesticated field-grown tepary bean in Arizona, USA, or with a commercial inoculant strain for Phaseolus vulgaris (CC511). Different degrees of plant reliance upon N₂ fixation for growth were generated by supplying the inoculated plants throughout growth with nutrients containing a range of concentrations of ¹⁵N-labeled NO₃ (0, 1, 2, 5 or 10 mM). An uninoculated treatment that received 10 mM ¹⁵N-labeled NO₃ was included to provide data for plants solely dependent upon NO₃ for growth. Six weeks after sowing, shoots were harvested for dry matter determination and subsequent ¹⁵N analysis, root-bleeding xylem sap was collected, and nodulation assessed. With regard to shoot biomass production, domesticated lines were more responsive to inoculation, but less responsive to applied N than wild types. All inoculated plants were nodulated, but the field isolates from tepary bean were more effective in N₂ fixation than strain CC511. It was concluded that tepary bean requires a specific inoculant to benefit from fixation of atmospheric N₂. Xylem sap samples were analysed for ureides (allantoin and allantoic acid), amino acid content (α-amino-N), and NO₃ concentration. The amount of ureide-N present in xylem sap was expressed as a percentage of total solute N, described as the relative abundance of ureide-N (RUN), for each N treatment and was compared to the proportion of plant N derived from N₂ fixation (%Ndfa) calculated using a ¹⁵N dilution technique. The RUN values ranged from 8% for saps collected from uninoculated plants provided with 10 mM NO₃ in the nutrient solution (%Ndfa=0) to 86-91% for nodulated plants grown in the absence of externally supplied NO₃ (%Ndfa=100). These data indicated that ureides were the principal product of N₂ fixation exported from the nodules to the shoot in xylem sap. Since RUN values were closely related to %Ndfa, it was proposed that N-solute analysis of xylem sap could provide a valuable analytical tool to monitor the symbiotic performance of tepary bean.     

Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

Can a mixed stand of N2-fixing and non-fixing plants restrict N2O emissions with increasing CO2 concentration?

Initial effects of elevated atmospheric CO₂ concentration on N₂O fluxes and biomass production of timothy/red clover were studied in the laboratory. The experimental design consisted of two levels of atmospheric CO₂ (ca. 360 and 720 μmol CO₂ mol⁻¹) and two N fertilisation levels (5 and 10 g N m⁻²). There was a total of 36 mesocosms comprising sandy loam soil, which were equally distributed in four thermo-controlled greenhouses. In two of the greenhouses, the CO₂ concentration was kept at ambient concentration and in the other two at doubled concentration. Forage was harvested and the plants fertilised three times during the basic experiment, followed by harvest, a fertilisation with the double amount of nitrogen and rise of water level. Under elevated CO₂, harvestable and total aboveground dry biomass production of a mixed Trifolium/Phleum stand was increased at both N treatments compared to ambient CO₂. The N₂O flux rates under ambient CO₂ were significantly higher at both N treatments during the early growth of mixed Phleum/Trifolium mesocosms compared to the N₂O flux rate under elevated CO₂. However, when the conditions were favourable for denitrification at the end of the experiment, i.e. N availability and soil moisture were high enough, the elevated CO₂ concentration enhanced the N₂O efflux.     

CH4 oxidation and emissions of CH4 and N2O from Lolium perenne swards under elevated atmospheric CO2

Emissions of N₂O and CH₄ and CH₄ oxidation rates were measured from Lolium perenne swards in a short-term study under ambient (36 Pa) and elevated (60 Pa) atmospheric CO₂ at the Free Air Carbon dioxide Enrichment experiment, Eschikon, Switzerland. Elevated pCO₂ increased (P<0.05) N₂O emissions from high N fertilised (11.2 g N m⁻²) swards by 69%, but had no significant effect on net emissions of CH₄. Application of ¹³C-CH₄ (11 μl l⁻¹; 11 at.% excess ¹³C) to closed chamber headspaces in microplots enabled determination of rates of ¹³C-CH₄ oxidation even when net CH₄ fluxes from main plots were positive. We found a significant interaction between fertiliser application rate and atmospheric pCO₂ on ¹³C-CH₄ oxidation rates that was attributed to differences in gross nitrification rates and C and N availability. CH₄ oxidation was slower and thought to be temporarily inhibited in the high N ambient pCO₂ sward. The most rapid CH₄ oxidation of 14.6 μg ¹³C-CH₄ m⁻² h⁻¹ was measured in the high fertilised elevated pCO₂ sward, and we concluded that either elevated pCO₂ had a stimulatory effect on CH₄ oxidation or inhibition of oxidation following fertiliser application was lowered under elevated pCO₂. Application of ¹⁴NH₄¹⁵NO₃ and ¹⁵NH₄¹⁵NO₃ (10 at.% excess ¹⁵N) to different replicates enabled determination of the respective contributions of nitrification and denitrification to N₂O emissions. Inhibition of CH₄ oxidation in the high fertilised ambient pCO₂ sward, due to competition between NH₃ and CH₄ for methane monooxygenase enzymes or toxic effects of NH₂OH or NO₂⁻ produced during nitrification, was hypothesised to increase gross nitrification (12.0 mg N kg dry soil⁻¹) and N₂O emissions during nitrification (327 mg ¹⁵N-N₂O m⁻² over 11 d). Our results indicate that increasing atmospheric concentrations of CO₂ may increase emissions of N₂O by denitrification, lower nitrification rates and either increase or decrease the ability of soil to act as a sink for atmospheric CH₄ depending on fertiliser management.